Field of the Invention
The present invention relates to the microbiological industry, and specifically to a coryneform bacterium which efficiently produces a heterologous fusion protein by secretory production and a method for secretory production of a heterologous fusion protein using the modified coryneform bacterium.
Brief Description of the Related Art
Inteins are proteins which are capable of catalyzing their own excision from a precursor protein with the concomitant joining of the flanking protein sequences, known as exteins (Vila-Perelló M. and Muir T. W., Biological applications of protein splicing, Cell, 2010, 143(2):191-200). An intermediate forms during the intein-mediated protein splicing, which can be further modified chemically to obtain a recombinant protein or a peptide. In particular, the intermediate can be modified to produce proteins which are ligated, circularized, or conjugated with radioisotopes or chemical drugs. Antibodies can also be site-specifically modified using intein-mediated protein splicing. Various kinds of post-translational modifications of antibodies such as conjugation with polyethylene glycol (PEG), toxins, or radioisotopes have been investigated for the purpose of enhancing the efficacy of the antibody drugs, or adding novel functions to the antibody drugs. For example, certolizumab pegol (Cimzia, UCB), a tumor-necrosis factor blocker which is a Fab-PEG conjugate, was approved by the FDA in 2008 for the treatment of adult patients with moderate-to-severe Crohn's disease. Conjugation with PEG may enhance plasma half-life. Another example of modification of antibodies is a conjugation of an antibody or an antibody-related molecule with a toxin moiety (so called antibody-drug conjugates, ADC).
A method for protein engineering known as “expressed protein ligation” has been developed which is suitable for modification of the C-terminus of a target protein of any size by a nucleophilic group-containing compounds (Muir T. W. et al., Expressed protein ligation: A general method for protein engineering, Proc. Natl. Acad. Sci. USA, 1998, 95:6705-6710). The method of “expressed protein ligation” was successfully applied to modification of the protein tyrosine kinase C-terminal Src kinase (Csk) at the C-terminus through the expression of a fusion protein and replacement of the intein with a synthetic phosphotyrosine peptide under thiolysis conditions. The method for ligation of the expressed proteins has been disclosed, which utilizes inteins such as the R1R1 intein from Methanobacterium thermotrophicum (U.S. Pat. No. 6,849,428 B1; Evans T. C. Jr. et al., The in vitro ligation of bacterially expressed proteins using an intein from Methanobacterium thermoautotrophicum, J. Biol. Chem., 1999, 274(7):3923-3926). The method for amidation of recombinant peptides expressed as fusion proteins containing inteins has also been disclosed (Cottingham I. R. et al., A method for the amidation of recombinant peptides expressed as intein fusion proteins in Escherichia coli, Nat. Biotechnol., 2001, 19:974-977). Most of the commercial pharmaceutical peptides are amidated at their C-terminus to prolong the half-life in vivo. The peptide amidation technology utilizing a combination of expression of the recombinant fusion proteins and intein-mediated amidation reaction is also an important technology for obtaining C-terminal amidated peptides. The intein-mediated modifications can also be used for conjugation of proteins with labeled compounds such as radioisotopes or fluorescent compounds, biotin, etc. (Mohlmann S. et al., Site-specific modification of ED-B-targeting antibody using intein-fusion technology, BMC Biotechnol., 2011, 11:76) and circularization of proteins (Sudheer P. D. et at., Cyclization tag for the detection and facile purification of backbone-cyclized proteins, Anal. Biochem., 2013, 436:137-141).
To facilitate intein-mediated protein modification, the target protein may be expressed as a fusion protein with an appropriate intein. In general, the target protein-intein fusion proteins are expressed in Escherichia coli. The IMPACT system from New England Biolabs (catalog No. N6951S) utilizing the pTWIN vector is the most published commercial intein system in E. coli. Formation of an insoluble inclusion body often occurs when the target protein-intein fusion protein is expressed in E. coli. Therefore, a refolding process may be necessary to obtain the active form of the target protein-intein fusion protein recovered from the insoluble fraction. Expression of a target protein-intein fusion protein into a culture medium is more favorable due to the convenience for recovering the folded target protein-intein fusion protein from the culture medium. Expression of a target protein-intein fusion protein into culture media has been demonstrated using a mammalian cell expression system (Mohlmann S. et al., Site-specific modification of ED-B-targeting antibody using intein-fusion technology, BMC Biotechnol., 2011, 11:76).
Methods for obtaining fused target proteins from the culture supernatant using bacterial expression systems are known. For, example, a method for expression and secretion of the Vα, Vβ and single chain Vα-Vβ fragments of murine T-cell receptors fused with a signal peptide was reported for Gram-negative bacteria cells (U.S. Pat. No. 6,399,368 B1). A chimeric pro-region from Streptomyces cinnamoneus transglutaminase was used to attain high level expression of Streptomyces mobaraensis transglutaminase as a fusion protein in Corynebacterium glutamicum (Date M. et al., J. Biotechnol., 2004, 110(3):219-226). A method for the extracellular production of target proteins fused with an outer membrane protein OmpF and an oligopeptide for cleavage by a proteolytic enzyme utilizing E. coli as an expression host is known (U.S. Pat. No. 7,491,528 B2). Signal sequences from genes of Pseudomonas fluorescens and Bacillus coagulans for efficient secretion of correctly folded proteins from Gram-negative bacteria as expression hosts have been described (EP 2108047 B1). Also, a process for the secretory production of an exogenous protein by using a coryneform bacterium has been disclosed (WO 2001023591 A1). A target exogenous protein was expressed and excreted by a coryneform bacterium to a culture medium as a fusion protein with a signal peptide. The target protein was obtained by treating the extracellularly released fusion protein with a protease to cleave and eliminate the pro-part (WO 2001023591 A1).
Data demonstrating secretory production of the target protein-intein fusion proteins (also referred to as the heterologous fusion proteins as described in the present invention) using a coryneform bacterium has not been previously reported.